Abstract All organisms require robust methods for regulating expression of their genetic material. For the past five decades, transcriptional control has been the dominant paradigm by which regulation of gene expression has been described. More recently, however, it has become clear that regulation of pre-mRNA splicing can be an equally important regulatory mechanism. Whereas initial estimates suggested that only a small fraction of human genes would be alternatively spliced, recent evidence demonstrates that as many as 95% of all human genes are subject to this level of regulation. Nevertheless, the mechanisms by which this regulation is manifested have not been widely elucidated. To better understand how pre-mRNA splicing functions as a regulatory control point, we have chosen to examine the genetically tractable fission yeast, Schizosaccharomyces pombe. In many ways, splicing in S. pombe looks similar to splicing in higher eukaryotes. Introns have been identified in nearly half of all S. pombe genes, and single genes are interrupted by as many as 16 introns. The evolutionary retention of these introns suggested to us that splicing regulation might be a prevalent mechanism for gene regulation in S. pombe, making it a compelling system in which to undertake these experiments. Indeed, results of our initial experiments have borne out this hypothesis. Using a novel set of genome-wide tools that we developed, we have now identified distinct subsets of transcripts whose splicing is regulated in response to changing cellular environment. Strikingly, many of these regulated events mimic the paradigms that define alternative splicing in mammalian cells. The goals of our current work are to understand the trans-acting factors and cis-regulatory elements that are necessary for this regulation. Toward this end, we will employ a novel, high-throughput reverse genetic screen that we recently developed to identify the full complement of cellular proteins that are necessary for these regulated events. Similarly, we will use a forward genetic approach to identify all of the elements within these transcripts that are required for their regulation. The combination of these approaches should provide tremendous insights into the mechanisms by which this organism can regulate its gene expression via this pathway. Given the high level of conservation between splicing in S. pombe and humans, this work is likely to provide important insights into splicing regulation in higher eukaryotes. Moreover, because a significant number of human diseases are associated with mis-regulation of splicing, our understanding of the underlying parameters of this pathway should be of immediate clinical significance. PUBLIC HEALTH RELEVANCE: Project Narrative Pre-mRNA splicing is an essential component of gene expression for eukaryotic organisms. Recent evidence suggests that regulation of pre-mRNA splicing is an important mechanism by which organisms can regulate their gene expression under changing developmental or environmental states. We are using unicellular organisms to try to uncover the mechanism by which this pathway functions.